Extraction and immobilization in one step of two beta-glucosidases released from a yeast strain of Debaryomyces hansenii

An extracellular, constitutive, and nonglucose repressed beta-glucosidase f rom a yeast strain of Debaryomyces hansenii was purified and immobilized us ing a one-step procedure on hydroxyapatite (HTP). Analysis of purified enzy me gave two bands both on SDS gel electrophoresis, native gel electrophores is, and capillary electrophoresis. The two bands on SDS gels were positive for carbohydrate staining. Their apparent molecular mass was estimated to b e 122 and 96 kDa with carbohydrates, and 109 and 81 kDa after carbohydrate removal, respectively. Amino acid analysis of electroblotted bands revealed that the N-terminus was blocked in both cases. Gel slices corresponding to the two bands, as obtained after native gel electrophoresis, were found to be reactive when incubated separately with p-nitro-phensyl-beta-D-glucopyr anoside (pNPG) as substrate. The K-m of the two forms coeluted from HTP in the same fractions was 3.68 +/- 0.06 mM. The optimum pH was 5. The immobili zed enzyme exhibited a lower activity than the purified free enzymes, but b oth were much more stable than the enzymes in cell-free supernatant. The tw o enzyme isoforms in the mixture were only active against few glycosides wi th beta-linkage configuration. Since the HTP-bound enzyme was found to be a ctive, stable, easily separable from the substrate, and reusable, it could be potentially used in its immobilized form for the release of specific-bou nd aroma in wine and fruit juices. (C) 1999 Elsevier Science Inc.